RESUMO
While GLP-1 and its analogues are important pharmaceutical agents in the treatment of type 2 diabetes and obesity, their susceptibility to aggregate into amyloid fibrils poses a significant safety issue. Many factors may contribute to the aggregation propensity, including pH. While it is known that the monomeric structure of GLP-1 has a strong impact on primary nucleation, probing its diverse structural ensemble is challenging. Here, we investigated the monomer structural ensembles at pH 3, 4, and 7.5 using state-of-the-art computational methods in combination with experimental data. We found significant stabilization of ß-strand structures and destabilization of helical structures at lower pH, correlating with observed aggregation lag times, which are lower under these conditions. We further identified helical defects at pH 4, which led to the fastest observed aggregation, in agreement with our far-UV circular dichroism data. The detailed atomistic structures that result from the computational studies help to rationalize the experimental results on the aggregation propensity of GLP-1. This work provides a new insight into the pH-dependence of monomeric structural ensembles of GLP-1 and connects them to experimental observations.
Assuntos
Peptídeo 1 Semelhante ao Glucagon , Peptídeo 1 Semelhante ao Glucagon/química , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Concentração de Íons de Hidrogênio , Termodinâmica , Simulação de Dinâmica Molecular , Agregados ProteicosRESUMO
Knotted proteins are rare but important species, yet how their complex topologies affect their physical properties is not fully understood. Here we combine single molecule nanopore experiments and all-atom MD simulations to study the electric-field-driven unfolding during the translocation through a model pore of individual protein knots important for methylating tRNA. One of these knots shows an unusual behavior that resembles the behavior of electrons hopping between two potential surfaces: as the electric potential driving the translocation reaction is increased, the rate eventually plateaus or slows back down in the "Marcus inverted regime". Our results shed light on the influence of topology in knotted proteins on their forced translocation through a pore connecting two electrostatic potential wells.
Assuntos
Conformação Proteica , Proteínas , Proteínas/químicaRESUMO
De novo protein design methods can create proteins with folds not yet seen in nature. These methods largely focus on optimizing the compatibility between the designed sequence and the intended conformation, without explicit consideration of protein folding pathways. Deeply knotted proteins, whose topologies may introduce substantial barriers to folding, thus represent an interesting test case for protein design. Here we report our attempts to design proteins with trefoil (31) and pentafoil (51) knotted topologies. We extended previously described algorithms for tandem repeat protein design in order to construct deeply knotted backbones and matching designed repeat sequences (N = 3 repeats for the trefoil and N = 5 for the pentafoil). We confirmed the intended conformation for the trefoil design by X ray crystallography, and we report here on this protein's structure, stability, and folding behaviour. The pentafoil design misfolded into an asymmetric structure (despite a 5-fold symmetric sequence); two of the four repeat-repeat units matched the designed backbone while the other two diverged to form local contacts, leading to a trefoil rather than pentafoil knotted topology. Our results also provide insights into the folding of knotted proteins.
Assuntos
Dobramento de Proteína , Proteínas , Conformação Proteica , Proteínas/genética , Proteínas/química , Domínios Proteicos , Sequências de Repetição em Tandem/genéticaRESUMO
The caseinolytic protease complex ClpXP is an important house-keeping enzyme in prokaryotes charged with the removal and degradation of misfolded and aggregated proteins and performing regulatory proteolysis. Dysregulation of its function, particularly by inhibition or allosteric activation of the proteolytic core ClpP, has proven to be a promising strategy to reduce virulence and eradicate persistent bacterial infections. Here, we report a rational drug-design approach to identify macrocyclic peptides which increase proteolysis by ClpP. This work expands the understanding of ClpP dynamics and sheds light on the conformational control exerted by its binding partner, the chaperone ClpX, by means of a chemical approach. The identified macrocyclic peptide ligands may, in the future, serve as a starting point for the development of ClpP activators for antibacterial applications.
RESUMO
The physical stability of peptide-based drugs is of great interest to the pharmaceutical industry. Glucagon-like peptide 1 (GLP-1) is a 31-amino acid peptide hormone, the analogs of which are frequently used in the treatment of type 2 diabetes. We investigated the physical stability of GLP-1 and its C-terminal amide derivative, GLP-1-Am, both of which aggregate into amyloid fibrils. While off-pathway oligomers have been proposed to explain the unusual aggregation kinetics observed previously for GLP-1 under specific conditions, these oligomers have not been studied in any detail. Such states are important as they may represent potential sources of cytotoxicity and immunogenicity. Here, we identified and isolated stable, low-molecular-weight oligomers of GLP-1 and GLP-1-Am, using size-exclusion chromatography. Under the conditions studied, isolated oligomers were shown to be resistant to fibrillation or dissociation. These oligomers contain between two and five polypeptide chains and they have a highly disordered structure as indicated by a variety of spectroscopic techniques. They are highly stable with respect to time, temperature, or agitation despite their noncovalent character, which was established using liquid chromatography-mass spectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results provide evidence of stable, low-molecular-weight oligomers that are formed by an off-pathway mechanism which competes with amyloid fibril formation.
Assuntos
Diabetes Mellitus Tipo 2 , Peptídeo 1 Semelhante ao Glucagon , Humanos , Peptídeos , Amiloide/química , Cromatografia em Gel , Fragmentos de Peptídeos/química , Peptídeos beta-Amiloides/químicaRESUMO
Antibody-drug conjugates (ADCs) are valuable therapeutic entities which leverage the specificity of antibodies to selectively deliver cytotoxins to antigen-expressing targets such as cancer cells. However, current methods for their construction still suffer from a number of shortcomings. For instance, using a single modification technology to modulate the drug-to-antibody ratio (DAR) in integer increments while maintaining homogeneity and stability remains exceptionally challenging. Herein, we report a novel method for the generation of antibody conjugates with modular cargo loading from native antibodies. Our approach relies on a new class of disulfide rebridging linkers, which can react with eight cysteine residues, thereby effecting all-in-one bridging of all four interchain disulfides in an IgG1 antibody with a single linker molecule. Modification of the antibody with the linker in a 1 : 1 ratio enabled the modulation of cargo loading in a quick and selective manner through derivatization of the linker with varying numbers of payload attachment handles to allow for attachment of either 1, 2, 3 or 4 payloads (fluorescent dyes or cytotoxins). Assessment of the biological activity of these conjugates demonstrated their exceptional stability in human plasma and utility for cell-selective cytotoxin delivery or imaging/diagnostic applications.
RESUMO
Although monoclonal antibodies have greatly improved cancer therapy, they can trigger side effects due to on-target, off-tumor toxicity. Over the past decade, strategies have emerged to successfully mask the antigen-binding site of antibodies, such that they are only activated at the relevant site, for example, after proteolytic cleavage. However, the methods for designing an ideal affinity-based mask and what parameters are important are not yet well understood. Here, we undertook mechanistic studies using three masks with different properties and identified four critical factors: binding site and affinity, as well as association and dissociation rate constants, which also played an important role. HDX-MS was used to identify the location of binding sites on the antibody, which were subsequently validated by obtaining a high-resolution crystal structure for one of the mask-antibody complexes. These findings will inform future designs of optimal affinity-based masks for antibodies and other therapeutic proteins.
Assuntos
Anticorpos Monoclonais , Anticorpos Monoclonais/química , Afinidade de Anticorpos , Sítios de LigaçãoRESUMO
There is an increasing emphasis on the critical evaluation of interbatch purity and physical stability of therapeutic peptides. This is due to concerns over the impact that product- and process-related impurities may have on safety and efficacy of this class of drug. Aspartic acid isomerization to isoaspartic acid is a common isobaric impurity that can be very difficult to identify without first synthesizing isoAsp peptide standards for comparison by chromatography. As such, analytical tools that can determine if an Asp residue has isomerized, as well as the site of isomerization within the peptide sequence, are highly sought after. Ion mobility-mass spectrometry is a conformation-selective method that has developed rapidly in recent years particularly with the commercialization of traveling wave ion mobility instruments. This study employed a cyclic ion mobility (cIMS) mass spectrometry system to investigate the conformational characteristics of a therapeutic peptide and three synthetic isomeric forms, each with a single Asp residue isomerized to isoAsp. cIMS was able to not only show distinct conformational differences between each peptide but crucially, in conjunction with a simple workflow for comparing ion mobility data, it correctly located which Asp residue in each peptide had isomerized to isoAsp. This work highlights the value of cIMS as a potential screening tool in the analysis of therapeutic peptides prone to the formation of isoAsp impurities.
Assuntos
Ácido Aspártico , Peptídeos , Ácido Aspártico/química , Cromatografia Líquida de Alta Pressão/métodos , Isomerismo , Espectrometria de Massas/métodos , Peptídeos/químicaRESUMO
14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid ß (Aß) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer's and Parkinson's diseases, respectively, a process that is intimately linked to the diseases' progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aß (Aß40) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of 15N-labeled Aß40 and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aß40 (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt ß-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aß40 and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.
Assuntos
Proteínas 14-3-3/metabolismo , Peptídeos beta-Amiloides/metabolismo , alfa-Sinucleína/metabolismo , Proteínas 14-3-3/fisiologia , Amiloide/metabolismo , Amiloide/fisiologia , Peptídeos beta-Amiloides/fisiologia , Humanos , Chaperonas Moleculares/fisiologia , Agregados Proteicos , Ligação Proteica/fisiologia , Conformação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas/fisiologia , Desdobramento de Proteína , alfa-Sinucleína/fisiologiaRESUMO
Antibody-drug conjugates have become one of the most actively developed classes of drugs in recent years. Their great potential comes from combining the strengths of large and small molecule therapeutics: the exquisite specificity of antibodies and the highly potent nature of cytotoxic compounds. More recently, the approach of engineering antibody-drug conjugate scaffolds to achieve highly controlled drug to antibody ratios has focused on substituting or inserting cysteines to facilitate site-specific conjugation. Herein, we characterize an antibody scaffold engineered with an inserted cysteine that formed an unexpected disulfide bridge during manufacture. A combination of mass spectrometry and biophysical techniques have been used to understand how the additional disulfide bridge forms, interconverts, and changes the stability and structural dynamics of the antibody intermediate. This quantitative and structurally resolved model of the local and global changes in structure and dynamics associated with the engineering and subsequent disulfide-bonded variant can assist future engineering strategies.
Assuntos
Especificidade de Anticorpos , Antineoplásicos/química , Imunoconjugados , Compostos de Sulfidrila/química , Anticorpos Monoclonais , Sítios de Ligação , Desenho de Fármacos , Modelos Moleculares , Conformação ProteicaRESUMO
Knots in proteins are hypothesized to make them resistant to enzymatic degradation by ATP-dependent proteases and recent studies have shown that whereas ClpXP can easily degrade a protein with a shallow 31 knot, it cannot degrade 52-knotted proteins if degradation is initiated at the C-terminus. Here, we present detailed studies of the degradation of both 31- and 52-knotted proteins by ClpXP using numerous constructs where proteins are tagged for degradation at both N- and C-termini. Our results confirm and extend earlier work and show that ClpXP can easily degrade a deeply 31-knotted protein. In contrast to recently published work on the degradation of 52-knotted proteins, our results show that the ClpXP machinery can also easily degrade these proteins. However, the degradation depends critically on the location of the degradation tag and the local stability near the tag. Our results are consistent with mechanisms in which either the knot simply slips along the polypeptide chain and falls off the free terminus, or one in which the tightened knot enters the translocation pore of ClpXP. Results of experiments on knotted protein fusions with a highly stable domain show partial degradation and the formation of degradation intermediates.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Escherichia coli/genética , Cinética , Proteólise , ATPases Associadas a Diversas Atividades Celulares/química , Endopeptidase Clp , Escherichia coli/enzimologia , Proteínas de Escherichia coli , Modelos Moleculares , Dobramento de ProteínaRESUMO
As a key player of the protein quality control network of the cell, the molecular chaperone Hsp70 inhibits the aggregation of the amyloid protein tau. To date, the mechanism of this inhibition and the tau species targeted by Hsp70 remain unknown. This is partly due to the inherent difficulty of studying amyloid aggregates because of their heterogeneous and transient nature. Here, we used ensemble and single-molecule fluorescence measurements to dissect how Hsp70 counteracts the self-assembly process of the K18 ΔK280 tau variant. We found that Hsp70 blocks the early stages of tau aggregation by suppressing the formation of tau nuclei. Additionally, Hsp70 sequesters oligomers and mature tau fibrils with nanomolar affinity into a protective complex, efficiently neutralizing their ability to damage membranes and seed further tau aggregation. Our results provide novel insights into the molecular mechanisms by which the chaperone Hsp70 counteracts the formation, propagation, and toxicity of tau aggregates.
Assuntos
Proteínas de Choque Térmico HSP70/farmacologia , Agregação Patológica de Proteínas/tratamento farmacológico , Proteínas tau/antagonistas & inibidores , Amiloide/efeitos dos fármacos , Fluorescência , Humanos , Imagem Individual de MoléculaRESUMO
The number of biological therapeutic agents in the clinic and development pipeline has increased dramatically over the last decade and the number will undoubtedly continue to increase in the coming years. Despite this fact, there are considerable challenges in the development, production and formulation of such biologics particularly with respect to their physical stabilities. There are many cases where self-association to form either amorphous aggregates or highly structured fibrillar species limits their use. Here, we review the numerous factors that influence the physical stability of peptides including both intrinsic and external factors, wherever possible illustrating these with examples that are of therapeutic interest. The effects of sequence, concentration, pH, net charge, excipients, chemical degradation and modification, surfaces and interfaces, and impurities are all discussed. In addition, the effects of physical parameters such as pressure, temperature, agitation and lyophilization are described. We provide an overview of the structures of aggregates formed, as well as our current knowledge of the mechanisms for their formation.
RESUMO
Over the years, advances in experimental and computational methods have helped us to understand the role of thermodynamic, kinetic and active (chaperone-aided) effects in coordinating the folding steps required to achieving a knotted native state. Here, we review such developments by paying particular attention to the complementarity of experimental and computational studies. Key open issues that could be tackled with either or both approaches are finally pointed out.
Assuntos
Dobramento de Proteína , Proteínas/química , Conformação ProteicaRESUMO
An increasing number of proteins that contain topological knots have been identified over the past two decades; however, their folding mechanisms are still not well understood. UCH-L1 has a 52-knotted topology. Here, we constructed a series of variants that contain a single tryptophan at different locations along the polypeptide chain. A study of the thermodynamic properties of the variants shows that the structure of UCH-L1 is remarkably tolerant to incorporation of bulky tryptophan side chains. Comprehensive kinetic studies of the variants reveal that they fold via parallel pathways on which there are two intermediate states very similar to wild-type UCH-L1. The structures of the intermediate states do not change significantly with mutation and therefore occupy local minima on the energy landscape that have relatively narrow basins. The kinetic studies also establish that there are considerably more tertiary interactions in the intermediate states than results from previous NMR studies suggested. The two intermediates differ from each other in the extent to which tertiary interactions between the highly stable central ß-sheet and flanking α-helices and loop regions are formed. There is strong evidence that these states are aggregation prone. The transition states from both I1 and I2 to the native state are plastic and change with mutation and denaturant concentration. Previous studies indicated that the threading event that creates the 52 knot occurs during these steps, suggesting that there is a broad energy barrier consistent with the chain undergoing some searching of conformational space as the threading takes place.
Assuntos
Engenharia de Proteínas , Dobramento de Proteína , Triptofano , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/genética , Humanos , Cinética , Simulação de Dinâmica Molecular , Mutação , Multimerização Proteica , Redobramento de Proteína , Estrutura Quaternária de Proteína , Desdobramento de Proteína , TermodinâmicaRESUMO
Aggregation and amyloid fibril formation of peptides and proteins is a widespread phenomenon. It has serious implications in a range of areas from biotechnological and pharmaceutical applications to medical disorders. The aim of this study was to develop a better understanding of the mechanism of aggregation and amyloid fibrillation of an important pharmaceutical, human glucagon-like peptide-1 (GLP-1). GLP-1 is a 31-residue hormone peptide that plays an important role regulating blood glucose levels, analogues of which are used for treatment of type 2 diabetes. Amyloid fibril formation of GLP-1 was monitored using thioflavin T fluorescence as a function of peptide concentration between pH 7.5 and 8.2. Results from these studies establish that there is a highly unusual pH-induced switch in GLP-1 aggregation kinetics. At pH 8.2, the kinetics are consistent with a nucleation-polymerization mechanism for fibril formation. However, at pH 7.5, highly unusual kinetics are observed, where the lag time increases with increasing peptide concentration. We attribute this result to the formation of off-pathway species together with an initial slow, unimolecular step where monomer converts to a different monomeric form that forms on-pathway oligomers and ultimately fibrils. Estimation of the pKa values of all the ionizable groups in GLP-1 suggest it is the protonation/deprotonation of the N-terminus that is responsible for the switch with pH. In addition, a range of biophysical techniques were used to characterize (1) the start point of the aggregation reaction and (2) the structure and stability of the fibrils formed. These results show that the off-pathway species form under conditions where GLP-1 is most prone to form oligomers.
Assuntos
Peptídeo 1 Semelhante ao Glucagon/química , Agregados Proteicos , Sequência de Aminoácidos , Humanos , Concentração de Íons de Hidrogênio , Cinética , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
Spontaneous folding of a polypeptide chain into a knotted structure remains one of the most puzzling and fascinating features of protein folding. The folding of knotted proteins is on the timescale of minutes and thus hard to reproduce with atomistic simulations that have been able to reproduce features of ultrafast folding in great detail. Furthermore, it is generally not possible to control the topology of the unfolded state. Single-molecule force spectroscopy is an ideal tool for overcoming this problem: by variation of pulling directions, we controlled the knotting topology of the unfolded state of the 52-knotted protein ubiquitin C-terminal hydrolase isoenzyme L1 (UCH-L1) and have therefore been able to quantify the influence of knotting on its folding rate. Here, we provide direct evidence that a threading event associated with formation of either a 31 or 52 knot, or a step closely associated with it, significantly slows down the folding of UCH-L1. The results of the optical tweezers experiments highlight the complex nature of the folding pathway, many additional intermediate structures being detected that cannot be resolved by intrinsic fluorescence. Mechanical stretching of knotted proteins is also of importance for understanding the possible implications of knots in proteins for cellular degradation. Compared with a simple 31 knot, we measure a significantly larger size for the 52 knot in the unfolded state that can be further tightened with higher forces. Our results highlight the potential difficulties in degrading a 52 knot compared with a 31 knot.
Assuntos
Redobramento de Proteína , Desdobramento de Proteína , Ubiquitina Tiolesterase/química , Pinças Ópticas , Imagem Individual de MoléculaRESUMO
The human ubiquitin C-terminal hydrolase, UCH-L1, is an abundant neuronal deubiquitinase that is associated with Parkinson's disease. It contains a complex Gordian knot topology formed by the polypeptide chain alone. Using a combination of fluorescence-based kinetic measurements, we show that UCH-L1 has two distinct kinetic folding intermediates that are transiently populated on parallel pathways between the denatured and native states. NMR hydrogen-deuterium exchange (HDX) experiments indicate the presence of partially unfolded forms (PUFs) of UCH-L1 under native conditions. HDX measurements as a function of urea concentration were used to establish the structure of the PUFs and pulse-labelled HDX NMR was used to show that the PUFs and the folding intermediates are likely the same species. In both cases, a similar stable core encompassing most of the central ß-sheet is highly structured and α-helix 3, which is partially formed, packs against it. In contrast to the stable ß-sheet core, the peripheral α-helices display significant local fluctuations leading to rapid exchange. The results also suggest that the main difference between the two kinetic intermediates is structure and packing of α-helices 3 and 7 and the degree of structure in ß-strand 5. Together, the fluorescence and NMR results establish that UCH-L1 neither folds through a continuum of pathways nor by a single discrete pathway. Its folding is complex, the ß-sheet core forms early and is present in both intermediate states, and the rate-limiting step which is likely to involve the threading of the chain to form the 52-knot occurs late on the folding pathway.
Assuntos
Ubiquitina Tiolesterase/metabolismo , Deutério/metabolismo , Medição da Troca de Deutério/métodos , Humanos , Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética/métodos , Conformação Proteica em alfa-Hélice/fisiologia , Conformação Proteica em Folha beta/fisiologia , Desnaturação Proteica , Dobramento de ProteínaRESUMO
We have investigated the potential role of molecular chaperones as modulators of the immune response by using α-synuclein (αSyn) as an aggregation-prone model protein. We first performed an in vitro immunoscreening with 21 preselected candidate chaperones and selected 2 from this set as displaying immunological activity with differential profiles, Grp94/Gp96 and FKBP4/52. We then immunized mice with both chaperone/α-synuclein combinations using monomeric or oligomeric α-synuclein (MαSyn or OαSyn, respectively), and we characterized the immune response generated in each case. We found that Grp94 promoted αSyn-specific T-helper (Th)1/Th17 and IgG1 antibody responses (up to a 3-fold increase) with MαSyn and OαSyn, respectively, coupled to a Th2-type general phenotype (generating 2.5-fold higher IgG1/IgG2 levels). In addition, we observed that FKBP4 favored a Th1-skewed phenotype with MαSyn but strongly supported a Th2-type phenotype with OαSyn (with a 3-fold higher IL-10/IFN-γ serum levels). Importantly, results from adoptive transfer of splenocytes from immunized animals in a Parkinson's disease mouse model indicates that these effects are robust, stable in time, and physiologically relevant. Taken together, Grp94 and FKBP4 are able to generate differential immune responses to α-synuclein-based immunizations, depending both on the nature of the chaperone and on the aggregation state of α-synuclein. Our work reveals that several chaperones are potential modulators of the immune response and suggests that different chaperones could be exploited to redirect the amyloid-elicited immunity both for basic studies of the immunological processes associated with neurodegeneration and for immunotherapy of pathologies associated with protein misfolding and aggregation.
Assuntos
Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/fisiologia , Proteínas de Ligação a Tacrolimo/metabolismo , alfa-Sinucleína/metabolismo , Imunidade Adaptativa , Animais , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Imunidade Inata , Masculino , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dobramento de Proteína , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/imunologia , alfa-Sinucleína/genéticaRESUMO
Understanding the mechanism by which a polypeptide chain thread itself spontaneously to attain a knotted conformation has been a major challenge in the field of protein folding. HP0242 is a homodimeric protein from Helicobacter pylori with intertwined helices to form a unique pseudo-knotted folding topology. A tandem HP0242 repeat has been constructed to become the first engineered trefoil-knotted protein. Its small size renders it a model system for computational analyses to examine its folding and knotting pathways. Here we report a multi-parametric study on the folding stability and kinetics of a library of HP0242 variants, including the trefoil-knotted tandem HP0242 repeat, using far-UV circular dichroism and fluorescence spectroscopy. Equilibrium chemical denaturation of HP0242 variants shows the presence of highly populated dimeric and structurally heterogeneous folding intermediates. Such equilibrium folding intermediates retain significant amount of helical structures except those at the N- and C-terminal regions in the native structure. Stopped-flow fluorescence measurements of HP0242 variants show that spontaneous refolding into knotted structures can be achieved within seconds, which is several orders of magnitude faster than previously observed for other knotted proteins. Nevertheless, the complex chevron plots indicate that HP0242 variants are prone to misfold into kinetic traps, leading to severely rolled-over refolding arms. The experimental observations are in general agreement with the previously reported molecular dynamics simulations. Based on our results, kinetic folding pathways are proposed to qualitatively describe the complex folding processes of HP0242 variants.